Antimicrobial Peptides (AMPs) are an essential component of the defense system of organisms throughout nature and offer protection against invading pathogens. AMPs do not target single defined molecular structures (epitopes), but act on the cell membrane, thus killing bacteria and fungi rapidly. Therefore, as opposed to conventional antibiotics, they are effective regardless of the metabolic activity of bacteria. In addition to their direct microbicidal activities, antimicrobial peptides are particularly attractive as certain peptides show multiple activities such as the regulation of the innate and adaptive immune systems, inflammation and wound healing, and additional antifungal, antiviral, antiparasitic and anticancerous activities. AMPs are quite diverse in sequence and secondary structure, but share some common properties. They are usually short (about 15 to 40 amino acids), cationic, amphipathic and exert their microbicidal effect mostly by compromising the bacterial membrane integrity. Interaction of AMPs with the anionic membrane surface of the target microbes leads to membrane permeabilization, cell lysis and death. It is generally accepted that the cytoplasmic membrane is the main target of most antimicrobial peptides, whereby accumulation of peptide in the membrane causes increased permeability and loss of barrier function, resulting in leakage of cytoplasmic components and cell death. Various molecular mechanisms for membrane permeabilization, some phenomenological and others more quantitative, have been proposed to explain the action of AMPs.
Experimental observations in model systems were mainly rationalized by the carpet or pore model (FIG. 1). In the carpet model, AMPs accumulate on the membrane surface oriented in a parallel fashion to the membrane, resulting in local membrane thinning and destabilization of the cell membrane leading to the release of intracellular content. However, there is compelling evidence that many AMPs also function in a detergent-like manner, by disrupting the packing and organization of the lipids in a nonspecific way (e.g., lipid clustering or segregation of polar and nonpolar groups of the lipids) or by inducing non-bilayer lipid aggregates. Moreover, some AMPs pass the cell membrane and interact with an intracellular target (FIG. 1), leading to loss of bacterial/fungal viability.
Clearly, the mode(s) of action of AMPs differ from those of conventional antibiotics, which often have simple targets, such as a unique epitope on the cell wall, or in the protein and RNA synthesis processes, allowing the pathogenic bacteria to develop resistance more rapidly. A major advantage of antimicrobial peptides over conventional antibiotics is that microbial resistance against these AMPs does not readily develop, most likely because these peptides—in contrast to conventional antibiotics—do not target single defined molecular structures (epitopes), but act on the cell membrane, thus killing bacteria and fungi within minutes. Thus, owing to the fast killing rate, being faster than the growth rate of bacteria, and nature of the target (substantial modification of the lipid composition would affect bacterial cell viability), resistance development is less likely. The emergence of mutants being resistant to AMPs has been determined by monitoring bacterial susceptibility after repeated sub-culturing in the presence of sub-inhibitory concentrations of the peptides, showing that the mutation rate was lower than other clinical antibiotics tested (e.g., ciprofloxacin and erythromycin). While the Minimal Inhibiting Concentration (MIC) of those antibiotics increased through all the subcultures (up to 64 times), the pressure of the peptides did not increase the MIC of the strain. Thus, in contrast to conventional antibiotics, resistance development in the presence of AMPs is less unlikely to occur. Furthermore, AMPs are fast-acting and biodegradable, which alleviates the current concern about residual antibiotics in the environment.
A wide variety of microbial infections are associated with biofilm formation, where microorganisms aggregate in a structured community in a self-produced polymeric matrix and adhere to a surface. An additional disadvantage of conventional antibiotics is that they do not ensure eradication of biofilm infections for the following reasons:
1) Insufficient penetration of conventional antibiotics into biofilms: The matrix in which bacteria are embedded protects them from external influences, such as antimicrobial substances. Most antibiotics are able to penetrate the biofilm, but their diffusion into the biofilm is slow so that they are inactivated before they can elicit their desired effect.
2) Low metabolic activity of bacteria: Biofilm-associated infections (BAI) are usually treated with vancomycin, often in combination with rifampicin. Although vancomycin is known to penetrate biofilms rather well—albeit at a significantly reduced transport rate—it poorly reduces the number of bacteria residing within the biofilm. Treatment with this antibiotic still has, therefore, a relatively high rate of failure, which can be explained by the low metabolic activity of bacteria in the biofilm, rendering the antibiotic ineffective.
3) Inactivation or degradation of the antibiotics: In BAI, antibiotics are mostly administered systemically. Therefore, they are prone to be removed from the bloodstream by renal clearance and degraded enzymatically in the blood and surrounding tissues. Enzymes (produced by bacteria) can directly destruct or modify the compound. These mechanisms actively reduce the concentration of drugs in the local environment. In biofilms, the low penetration poses an additional problem. Increasing the systemically administered concentration is not feasible due to the toxicity of high blood concentrations of antibiotics.
4) Bacteria have developed resistance: On top of the general increase of bacterial resistance to antibiotics, due to the decreased concentrations of antibiotics in the deeper layers, the risk that bacteria escape from antibiotic pressure is higher, which may lead to the survival of mutants that have increased resistance to these antibiotics. It has even been reported that suboptimal concentrations of antibiotics, including vancomycin, enhance biofilm formation. Moreover, repetitive administration of conventional antibiotics that have an insufficient effect promotes the development of antibiotic resistance.
5) Conventional antibiotics cause the release of pro-inflammatory microbial compounds: It has been shown that in BAI, peri-implant tissue is colonized by bacteria. To a large extent, this is due to the deregulation of the local immune response. This is the reason why, in many cases, the infection persists, even after replacement of the implant. Implantation of a biomaterial provokes an inflammatory response known as the “foreign body response,” characterized by sequential influx of neutrophils, macrophages/monocytes and lymphocytes, followed by fusion of macrophages to multinucleated foreign body giant cells lining the biomaterial, novel fibroblast foiination and deposition of fibrin, leading to fibrosis/encapsulation of the foreign body. This sequence of events is highly regulated by molecular signals such as cytokines produced by the cell types involved. In case of infection, the host immune system is additionally triggered by molecules of the bacteria designated as “Pathogen-Associated Molecular Patterns” (PAMPs) recognized by specific receptors on the host cells, such as Toll-Like Receptors (TLRs). For example, bacterial peptidoglycan or lipopolysaccharide are recognized by TLR2 and TLR4, and are potent inducers of inflammatory responses. The activation of the immune system, both by the foreign body response and the bacterial infection, leads to an “over-the-top” reaction of the host immune system, leading to inflamed and disrupted tissue; in fact, providing the ideal environment for infection. Thus, the simultaneous activation by biomaterial and PAMPs can have deleterious effects on immune function and strongly increase susceptibility to infection.
At present, over 2,000 different antimicrobial peptide sequences are known (see, for instance, the World Wide Web at bbcm.univ.trieste.itttossi/search.htm), including cecropins, defensins, magainins and cathelicidins. Antimicrobial peptides and proteins are, for instance, described in:
U.S. Pat. No. 6,503,881, which discloses cationic peptides being an indolicidin analogue to be used as an antimicrobial peptide. The cationic peptides are derived from different species, including animals and plants.
U.S. Pat. No. 5,912,230, which discloses anti-fungal and anti-bacterial histatin-based peptides and methods for treatment of fungal and bacterial infections. The peptides are based on defined portions of the amino acid sequences of naturally occurring human histatins.
U.S. Pat. No. 5,717,064, which discloses methylated lysine-rich lytic peptides. The lytic peptides are tryptic digestion resistant and non-natural. The lytic peptides are suitable for in vivo administration.
U.S. Pat. No. 5,646,014, which discloses an antimicrobial peptide isolated from an antimicrobial fraction from silkworm hemolymph. The peptide exhibits antimicrobial activity against several bacterial strains, such as Escherichia coli, Staphylococcus aureus and Bacillus cereus. 
WO 2004/016653, which discloses a peptide based on the 20 to 44 sequence of azurocidin. This peptide contains a loop structure linked by disulfide bridges.
U.S. Pat. No. 6,495,516, which discloses peptides based on the bactericidal 55 kDa protein bactericidal/permeability-increasing protein (BPI). The peptides exert antimicrobial effects as well as having LPS-neutralizing capacity.
WO 01/81578, which discloses numerous sequences encoding G-coupled protein receptor-related polypeptides, which may be used for numerous diseases.
WO 2004/067563 and WO 2005/040192, that disclose antimicrobial peptides based on peptide LL-37, the 37 C-terminal amino acid of the human cathelicidin.
Several AMPs, daptomycin and DPK-060, are currently in clinical use and/or development, e.g., polymyxin B, nisin, pexiganan, omiganan, iseganan. Further, up to phase 2 clinical trials have been performed for OP-145, a 24-amino acid peptide derived from the endogenous human cathelicidin antimicrobial peptide LL-37. OP-145 has been developed as an endotoxin-neutralizing antimicrobial peptide for the topical treatment of chronic otitis media. The currently known AMPs still have a few drawbacks. Although proteolytic degradation is beneficial for the environment (no residual AMPs), it prevents dynamic circulation. This is also caused by efficient peptide clearance. Also, the exact working mechanisms of AMPs remain largely unknown, so it is difficult to foresee their true applications and full potential. For example, it is often not known how AMPs interact with host cells to induce their effects. Therefore, the use of AMPs in clinical indications has been limited to topical applications.
Various bacteria, such as P. aeruginosa, E. faecalis, Proteus mirabilis, Streptococcus pyogenes and S. aureus all secrete proteases that degrade several antimicrobial peptides, such as the cathelicidin LL-37. Thus, protease-resistant antimicrobial peptides are advantageous from a therapeutical standpoint. Additionally, many of the antimicrobial peptides are not very efficient in challenging microorganisms such as bacteria, e.g., S. aureus and P. aeruginosa, frequently playing key roles in problematic pathogeneses, and need to be optimized to show an increased effect. Furthermore, due to potential lytic as well as other properties of AMPs against bacterial as well as mammalian membranes, one of the challenges in designing new peptides relies on developing AMPs with high specificity against microorganisms such as bacterial or fungal cells as compared to cellular membranes of the infected patient, i.e., a high therapeutic index (minimal hemolytic concentration/minimal antimicrobial activity; MHC/MEC).
Therefore, even though there are a relatively large number of antimicrobial peptides available today, there is still an increased need of new improved antimicrobial peptides, which can be used to counteract microbes, in particular, those that are resistant or tolerant against antibiotic agents and/or other antimicrobial agents. More importantly, there is a need for new antimicrobial peptides, which are non-allergenic when introduced into mammals such as human beings and that have high specificity against pathogenic microorganisms.